Homeostatic mini-intestines through scaffold-guided organoid morphogenesis.

Epithelial organoids, such as those derived from stem cells of the intestine, have great potential for modelling tissue and disease biology1-4. However, the approaches that are used at present to derive these organoids in three-dimensional matrices5,6 result in stochastically developing tissues with a closed, cystic architecture that restricts lifespan and size, limits experimental manipulation and prohibits homeostasis. Here, by using tissue engineering and the intrinsic self-organization properties of cells, we induce intestinal stem cells to form tube-shaped epithelia with an accessible lumen and a similar spatial arrangement of crypt- and villus-like domains to that in vivo. When connected to an external pumping system, the mini-gut tubes are perfusable; this allows the continuous removal of dead cells to prolong tissue lifespan by several weeks, and also enables the tubes to be colonized with microorganisms for modelling host-microorganism interactions. The mini-intestines include rare, specialized cell types that are seldom found in conventional organoids. They retain key physiological hallmarks of the intestine and have a notable capacity to regenerate. Our concept for extrinsically guiding the self-organization of stem cells into functional organoids-on-a-chip is broadly applicable and will enable the attainment of more physiologically relevant organoid shapes, sizes and functions.

Engineered signaling centers for the spatially controlled patterning of human pluripotent stem cells.

Signaling centers, localized groups of cells that secrete morphogens, play a key role in early development and organogenesis by orchestrating spatial cell fate patterning. Here we present a microfluidic approach that exposes human pluripotent stem cell (hPSC) colonies to spatiotemporally controlled morphogen gradients generated from artificial signaling centers. In response to a localized source of bone morphogenetic protein 4 (BMP4), hPSC colonies reproducibly break their intrinsic radial symmetry to produce distinct, axially arranged differentiation domains. Counteracting sources of the BMP antagonist NOGGIN enhance this spatial control of cell fate patterning. We also show how morphogen concentration and cell density affect the BMP response and germ layer patterning. These results demonstrate that the intrinsic capacity of stem cells for self-organization can be extrinsically controlled through the use of engineered signaling centers.

The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing.

During wound healing, the distribution, availability, and signaling of growth factors (GFs) are orchestrated by their binding to extracellular matrix components in the wound microenvironment. Extracellular matrix proteins have been shown to modulate angiogenesis and promote wound healing through GF binding. The hemostatic protein von Willebrand factor (VWF) released by endothelial cells (ECs) in plasma and in the subendothelial matrix has been shown to regulate angiogenesis; this function is relevant to patients in whom VWF deficiency or dysfunction is associated with vascular malformations. Here, we show that VWF deficiency in mice causes delayed wound healing accompanied by decreased angiogenesis and decreased amounts of angiogenic GFs in the wound. We show that in vitro VWF binds to several GFs, including vascular endothelial growth factor-A (VEGF-A) isoforms and platelet-derived growth factor-BB (PDGF-BB), mainly through the heparin-binding domain (HBD) within the VWF A1 domain. VWF also binds to VEGF-A and fibroblast growth factor-2 (FGF-2) in human plasma and colocalizes with VEGF-A in ECs. Incorporation of the VWF A1 HBD into fibrin matrices enables sequestration and slow release of incorporated GFs. In vivo, VWF A1 HBD-functionalized fibrin matrices increased angiogenesis and GF retention in VWF-deficient mice. Treatment of chronic skin wounds in diabetic mice with VEGF-A165 and PDGF-BB incorporated within VWF A1 HBD-functionalized fibrin matrices accelerated wound healing, with increased angiogenesis and smooth muscle cell proliferation. Therefore, the VWF A1 HBD can function as a GF reservoir, leading to effective angiogenesis and tissue regeneration.

[Visualization of Effectiveness of the Medical Safety Measures -Suggestion of the Evaluation Technique for Rare Medical Incident].

For events with a low occurrence rate, such as medical incidents, we were able to determine the evaluation before and after taking medical safety measures by statistical methods (testing for differences in population rate). The point of this method is that we evaluated the occurrence rate of incidents to the total number of examinations (number of incident occurrence real number plus number of examinations carried out without any problems). Our results suggest that this technique becomes the evaluation technique as the effective method of medical safety measures. The present studies demonstrated that the evaluation technique by the testing for differences in population rate become the indicator to judge the effectiveness of the medical safety measures in the following cases. (1) When we evaluate the decrease in incident for the long term before and after safety measures. (2) When we evaluate the effectiveness of measures in the middle evaluation after safety measures.

Defined three-dimensional microenvironments boost induction of pluripotency.

Since the discovery of induced pluripotent stem cells (iPSCs), numerous approaches have been explored to improve the original protocol, which is based on a two-dimensional (2D) cell-culture system. Surprisingly, nothing is known about the effect of a more biologically faithful 3D environment on somatic-cell reprogramming. Here, we report a systematic analysis of how reprogramming of somatic cells occurs within engineered 3D extracellular matrices. By modulating microenvironmental stiffness, degradability and biochemical composition, we have identified a previously unknown role for biophysical effectors in the promotion of iPSC generation. We find that the physical cell confinement imposed by the 3D microenvironment boosts reprogramming through an accelerated mesenchymal-to-epithelial transition and increased epigenetic remodelling. We conclude that 3D microenvironmental signals act synergistically with reprogramming transcription factors to increase somatic plasticity.

Preliminary study of luminescence phenomena from various materials under ultra-high dose rate proton beam irradiation for dose management.

This research aimed to identify materials capable of emitting visible light useful for dose management at ultra-high dose rate (uHDR). Various materials were irradiated with proton beams at a normal dose rate (NDR) and uHDR, and the resulting surface luminescence was captured using a high-sensitivity camera. The luminescence images were compared with the corresponding dose distributions. The luminescence of Tough Water Phantoms (Kyoto Kagaku Co. Ltd.) with various thicknesses was also observed to evaluate the depth distributions. Dose distributions were measured using two-dimensional ionization chamber detector arrays. The Tough Bone Phantom (Kyoto Kagaku Co. Ltd.) exhibited the strongest luminescence among the materials, followed by the Tough Water Phantom. The metals exhibited relatively weak luminescence. The luminescence profiles of the Tough Water Phantom, water, the Tough Lung Phantom (Kyoto Kagaku Co. Ltd.), and an acrylic were similar to the dose profiles. The luminescence distribution of the Tough Water Phantom in the depth direction was similar to that of the dose distributions. The luminescence at uHDR and NDR were approximately equivalent. The Tough Water Phantom was found to be a suitable material for dosimetry, even at uHDR. More detailed measurement data, such as wavelength data, must be collected to elucidate the luminescence mechanism.

Dose Evaluation in 2-Phase Method for Advanced Esophageal Cancer by Hybrid Irradiation Techniques.

Purpose: In concurrent chemoradiotherapy for advanced esophageal cancer, a 2-phase method consisting of initial irradiation of a wide elective nodal region and boost irradiation of the primary lesion is commonly employed. Although dose escalation to the primary lesion may be required to achieve higher local control rates, the radiation dose to critical organs must not exceed dose constraints. To achieve an optimum balance of dose prescription and dose reduction to surrounding organs, such as the lungs and heart, we compared hybrid dose distributions and investigated the best combination of the following recent irradiation techniques: volumetric modulation arc therapy (VMAT), proton broad-beam irradiation, and intensity-modulated proton beam therapy (IMPT). Materials and Methods: Forty-five patients with advanced esophageal cancer whose primary lesions were located in the middle- or lower-thoracic region were studied. Radiotherapy plans for the initial and boost irradiation in the 2-phase method were calculated using VMAT, proton broad-beam irradiation, and IMPT calculation codes, and the dose-volume histogram indices of the lungs and heart for the accumulated plans were compared. Results: In plans using boost proton irradiation with a prescribed dose of 60 Gy(RBE), all dose-volume histogram indices were significantly below the tolerance limits. Initial and boost irradiation with VMAT resulted in the median dose of V30 Gy(RBE)(heart) of 27.4% and an achievement rate below the tolerance limit of 57.8% (26 cases). In simulations of dose escalation up to 70 Gy(RBE), initial and boost IMPT resulted in the highest achievement rate, satisfying all dose constraints in 95.6% (43 cases). Conclusion: Applying VMAT to both initial and boost irradiation is not recommended because of the increased risk of the cardiac dose exceeding the tolerance limit. IMPT may allow dose escalation of up to 70 Gy(RBE) without radiation risks to the lungs and heart in the treatment of advanced esophageal cancer.

Multiscale microenvironmental perturbation of pluripotent stem cell fate and self-organization.

The combination of microfluidics with engineered three-dimensional (3D) matrices can bring new insights into the fate regulation of stem cells and their self-organization into organoids. Although there has been progress in 3D stem cell culturing, most existing in vitro methodologies do not allow for mimicking of the spatiotemporal heterogeneity of stimuli that drive morphogenetic processes in vivo. To address this, we present a perfusion-free microchip concept for the in vitro 3D perturbation of stem cell fate. Stem cells are encapsulated in a hydrogel compartment that is flanked by open reservoirs for the diffusion-driven generation of biomolecule gradients. Juxtaposing additional compartments bearing supportive cells enables investigating the influence of long range cell-cell communication. We explore the utility of the microchips in manipulating early fate choices and self-organizing characteristics of 3D-cultured mouse embryonic stem cells (mESCs) under neural differentiation conditions and exposure to gradients of leukemia inhibitory factor (LIF). mESCs respond to LIF gradients in a spatially dependent manner. At higher LIF concentrations, multicellular colonies maintain pluripotency in contrast, at lower concentrations, mESCs develop into apicobasally polarized epithelial cysts. This versatile system can help to systematically explore the role of multifactorial microenvironments in promoting self-patterning of various stem cell types.

Generation of Induced Pluripotent Stem Cells in Defined Three-Dimensional Hydrogels.

Since the groundbreaking discovery of induced pluripotent stem cells (iPSCs) many research groups have attempted to improve the efficiency of the classical cell reprogramming process. Surprisingly, the contribution of the three-dimensional (3D) microenvironment to iPSC generation has been largely overlooked. Here we describe a protocol for the generation of iPSCs in defined poly(ethylene glycol) (PEG)-based hydrogels that, besides allowing higher reprogramming efficiency, are also a powerful tool to study the influence of biophysical parameters on iPSC generation.

Decoding of position in the developing neural tube from antiparallel morphogen gradients.

Like many developing tissues, the vertebrate neural tube is patterned by antiparallel morphogen gradients. To understand how these inputs are interpreted, we measured morphogen signaling and target gene expression in mouse embryos and chick ex vivo assays. From these data, we derived and validated a characteristic decoding map that relates morphogen input to the positional identity of neural progenitors. Analysis of the observed responses indicates that the underlying interpretation strategy minimizes patterning errors in response to the joint input of noisy opposing gradients. We reverse-engineered a transcriptional network that provides a mechanistic basis for the observed cell fate decisions and accounts for the precision and dynamics of pattern formation. Together, our data link opposing gradient dynamics in a growing tissue to precise pattern formation.

Proliferation, morphology, and pluripotency of mouse induced pluripotent stem cells in three different types of alginate beads for mass production.

Induced pluripotent stem cells (iPSCs) are expected to be an ideal cell source for biomedical applications, but such applications usually require a large number of cells. Suspension culture of iPSC aggregates can offer high cell yields but sometimes results in excess aggregation or cell death by shear stress. Hydrogel-based microencapsulation can solve such problems observed in Suspension culture, but there is no systematic evaluation of the possible capsule formulations. In addition, their biological effects on entrapped cells are still poorly studied so far. We, therefore, immobilized mouse iPSCs in three different types of calcium-alginate (Alg-Ca) hydrogel-based microcapsules; (i) Alg-Ca capsules without further treatment (Naked), (ii) Alg-Ca capsules with poly-l-lysine (PLL) coating (Coated), and (iii) Alg-PLL membrane capsules with liquid cores (Hollow). After 10 days of culture within the medium containing serum and leukemia inhibitory factor, we obtained good cellular expansions (10-13-fold) in Coated and Hollow capsules that were similar to Suspension culture. However, 32 ± 9% of cellular leakage and lower cell yield (about threefold) were observed in Naked capsules. This was not observed in Coated and Hollow capsules. In addition, immunostaining and quantitative RT-PCR showed that the formation of primitive endodermal layers was suppressed in Coated capsules contrary to all other formulations. This agenesis of primitive endoderm layers in Coated capsules is likely to be the main cause of the significantly better pluripotency maintenance in hydrogel-based encapsulation culture. These results are helpful in further optimizing hydrogel-based iPSC culture, which can maintain better local cellular environments and be compatible with mass culture.

Development of bioactive hydrogel capsules for the 3D expansion of pluripotent stem cells in bioreactors.

Pluripotent stem cells hold great promise for many pharmaceutical and therapeutic applications. However, the lack of scalable methodologies to expand these cells to clinically relevant numbers is a major roadblock in realizing their full potential. To address this problem, we report here a scalable approach for the expansion of pluripotent stem cells within bioactive hydrogel capsules in stirred bioreactors. To achieve rapid crosslinking of cellular microenvironments with tuneable, cell-instructive functionality, we combined calcium-mediated alginate (CaAlg) complexation with crosslinking of poly(ethylene glycol) (PEG) macromers via a Michael-type addition. The resulting hybrid networks have been shown to have very good handling properties and can be readily decorated with biologically active signals such as integrin ligands or Cadherin-based motifs to influence the fate of mouse induced pluripotent stem (iPS) cells. Air-driven co-axial extrusion was used to reproducibly generate gel microcapsules in high-throughput. Furthermore, the gel capsules can be enveloped in a poly(l-lysine) shell to control swelling or molecular permeability independently of the gel composition. iPS cells entrapped within such capsules expanded with limited commitment to the endodermal lineage. Functionalization of gels with an appropriate density of Arg-Gly-Asp (RGD) ligands further increased the iPS cell expansion rate and reduced the spontaneous differentiation. Therefore, the combination of micro-scale instruction of cell fate by an engineered microenvironment and macro-scale cell manipulation in bioreactors opens up exciting opportunities for stem cell-based applications.